Purification and initial characterization of an enzyme with ...

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Biochem. J. (1987) 245, 831-841 (Printed in Great Britain)

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities Jack E. BALDWIN,* Robert M. ADLINGTON,* Janice B. COATES,* M. James C. CRABBE,* Nicholas P. CROUCH,* John W. KEEPING,* Graham C. KNIGHT,* Christopher J. SCHOFIELD,* Hong-Hoi TING,* Carlos A. VALLEJO,* Maureen THORNILEYt and Edward P. ABRAHAMt *Dyson Perrins Laboratory, University of Oxford, South Parks Road, Oxford OXI 3QY, and tThe Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OXI 3RE, U.K.

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40000) exhibited

a

pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+

as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable and for over 4 weeks at -70°C in the presence of mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A fl-sulphoxide and a 3,f-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction. Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3,8-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the 02

enzyme. were separated and partially purified by DEAE-Trisacryl chromatography (Jensen et al., 1985). In the eukaryote Cephalosporium acremonium the activities were not separated chromatographically on the same resin (Scheidegger et al., 1984), and it was suggested that they were associated with a single enzyme in this organism. In the present paper we describe methods for the assay and purification of an enzyme with both expandase and hydroxylase activities and some of the properties of the purified product. By analogy with the chemical conversion of penicillins into cephalosporins (Morin et al., 1963), the fl-sulphoxide (4) has been proposed as a potential intermediate in the biosynthetic transformation ofpenicillin N (1) into DAOC (2) (Baldwin et al., 1978; Queener & Neuss, 1982). Alternatively, the 8-methylene hydroxy penam (5) has also been proposed as a feasible intermediate for the bioconversion (Abraham, 1977; Baldwin et al., 1978), a transformation for which there is also a synthetic precedent (Stork & Cheung, 1965). The postulated intermediates are in stereochemical accord with the observation that the 3,-methyl group of penicillin N (1) is incorporated into the dihydrothiazine moiety of DAOC (2) (Kluender et al., 1973; Neuss et al., 1973;

INTRODUCTION Although the reactions catalysing the biosynthesis of penicillins and cephalosporins have been demonstrated in cell-free systems, it is only very recently that we have begun to understand the mechanisms of some of these reactions. The enzyme catalysing the cyclization of the tripeptide L-a-aminoadipoyl-L-cysteinyl-D-valine to form isopenicillin N (isopenicillin N synthetase) has been purified to homogeneity from Cephalosporium acremonium (Acremonium chrysogenum) (Pang et al., 1984; Hollander et al., 1984; Baldwin et al., 1985) and this enzyme cloned into Escherichia coli (Samson et al., 1985). In Cephalosporium and Streptomyces the L-a-aminoadipoyl side chain of isopenicillin N is epimerized to the D form, yielding penicillin N (1) (Jayatilake et al., 1981). This is further metabolized to cephalosporin C (for reviews see Abraham, 1985; Jensen, 1986). The fivemembered thiazolidine ring of penicillin N (1) undergoes an oxidative expansion ('expandase reaction') to a six-membered cephem [deacetoxycephalosporin C (DAOC) (2)], which in turn is hydroxylated to form deacetylcephalosporin C (DAC) (3) (Scheme 1). In the prokaryote Streptomyces clavuligerus the two activities

+

HNH3N N

oCOHN0 1

C02H

Hj

H3N

CH3 C02H

r

2

N

CH20H

3

Scheme 1. Abbrevations used: DAOC, deacetoxycephalosporin C; DAC, deacetylcephalosporin C; DTT, dithiothreitol; f.p.l.c., fast protein liquid chromatography. t Present address: Mars Ltd., Slough, Berks. SLI 4JX, U.K.

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J. E. Baldwin, R. M. Adlington, C. J. Schofield, N. P. Crouch & N. J. Turner, unpublished work). We therefore undertook the syntheses of compounds 4 and 5 in an attempt to evaluate these hypotheses. MATERIALS AND METHODS Assay of expandase and hydroxylase activities (a) Bioassay of expandase activity. Reaction mixtures contained DTT (1 mM), ascorbic acid (1 mM), 2oxoglutarate (0.8 mM). FeSO4 (0.04 mM), (NH4)2SO4 (100 mM) and penicillin N (0.14 mM) in 50 mM-Tris/HCl buffer, pH 7.4, in a final volume of 250 ,l. Mixtures were incubated at 27 'C in an orbital incubator, shaking at 250 rev./min. Samples were taken at 20, 40 and 60 min, and the reaction was stopped by the addition of an equal volume of methanol. After centrifugation, 100 ,1 samples of supernatants were loaded into wells in bioassay plates (Smith et al., 1967) containing supersensitive E. coli ESS 21/30 and fl-lactamase I (from Bacillus cereus) (10000 units/ml), to destroy unconverted penicillin N (1). Plates were incubated overnight at 37 'C. The diameters of the inhibition zones were measured, and amounts of product estimated by reference to standards of cephalosporin C. One unit of enzyme is that amount catalysing the production of 1 lsmol of product/min under the conditions used. DAOC (2) and DAC (3) produced identical zone sizes in the bioassay plates. Thus, although there was some conversion of DAOC into DAC in enzyme incubations budged by the h.p.l.c. assay (c)], the sum total of product (DAC + DAOC) reflected DAOC production in the expandase reaction.

(b) T.l.c./bioassay of hydroxylase activity. Reaction mixtures were as described above for (a), except that DAOC (2) (0.28 mM) was used as substrate and the final volume was 20 ,ul. Reaction mixtures were incubated at 27 'C for 60 min, when methanol (20 ,1) was added to stop the reaction, and the samples were centrifuged to remove protein. Samples of supernatants (4 1l) were loaded on to reverse-phase t.l.c. plates (Opti-Up C12; Fluka) and developed with 25 mM-NH4HCO3 for approx. 30 min to separate DAOC (2) and DAC (3). Plates were dried and blotted on to bioassay plates containing E. coli ESS 21/30 for 30 min at room temperature. After incubation of bioassay plates at 37 'C overnight, the position and size of inhibition zones relative to DAOC (2) (RF approx. 0.45) and DAC (3) (RF approx. 0.65) standards were recorded. This method could also be used to monitor expandase activity, by replacing DAOC (2) with penicillin N (1) as the substrate in the enzyme incubation, and using bioassay plates containing alactamase I (from B. cereus) as described in (a). (c) H.p.l.c. assays for expandase and hydroxylase activities. Incubations, with either penicillin N (1) or DAOC (2) as substrate, were as described above. Samples were taken at zero time and after 60 min. After removal of protein by filtration in the Centrifree system (Amicon Corp., Stonehouse, Glos., U.K.) (Petchey & Crabbe, 1984), samples (10-30 ,ul) were loaded on to a,uBondapak C18 h.p.l.c. column (Waters Associates, Milford, MA,

U.S.A.) and eluted isocratically with 25 mM-NH4HCO3 at 1 ml/min, with monitoring at 220 nm. In other

J. E. Baldwin and others

experiments protein was removed by precipitation with an equal volume of methanol, samples were loaded on to a,uBondapak C18 h.p.l.c. column, the products were eluted isocratically with 50 mM-sodium phosphate buffer (pH 3.8)/methanol/water (12:1:7, by vol.), and the eluate was monitored at 254 nm. In all incubations product formation was linear for up to 1 h, and initial velocity was proportional to enzyme concentration under the conditions used in these studies. Growth of organism C. acremonium C.O. 728 was grown in a chemically defined medium as previously described (Pang et al., 1984). Growth was monitored at 550 nm in a PyeUnicam SP6-550 spectrophotometer. Mycelia were harvested and stored as described previously (Pang et al., 1984). For the determination of the time course of enzyme production, enzyme activity was assessed after precipitation of expandase and hydroxylase from crude extracts with (NH4)2SO4 (Table 1). Antibiotic production was determined by bioassays with E. coli ESS and Staphylococcus aureus N.C.T.C. 6571 as test organisms. Preparation of cell-free extracts These were prepared in a Dyno-Mill grinder (Glen Creston, Stanmore, Middx., U.K.) essentially as described previously (Pang et al., 1984), by using the continuous-flow method and 50 mM-Tris/HCl buffer, pH 7.4, containing DTT (2 mM) and NaN3 (0.015%). Amino acid analysis Thrice-distilled HCI (5.7 M) was used for hydrolysis, samples were treated by phenyl isothiocyanate labelling and analysed on a Waters Pico-Tag amino acid analysis h.p.l.c. (reverse-phase) system and 840 data-control station with Expert software (revision 4).

SDS/polyacrylamide-gel electrophoresis This was performed by the method of Laemmli (1970), in a slab-gel apparatus (Bio-Rad Laboratories, Richmond, CA, U.S.A.) with 10% (w/v) polyacrylamide gels. Ovotransferrin (hen's-egg; 76-78 kDa), albumin (bovine serum; 66.2 kDa), ovalbumin (hen's-egg; 45 kDa), carbonic anhydrase (bovine erythrocyte; 30 kDa), myoglobin (horse; 17.2 kDa) and cytochrome c (horse; 12.3 kDa) were used as molecular-mass markers (BDH Chemicals, Poole, Dorset, U.K.). Chromatography Matrex Gel Red A (Procion Red HE3B-agarose) was

obtained from Amicon Corp. Buffers for f.p.l.c. (Pharmacia, Milton Keynes, Bucks., U.K.) were made up in Milli-Q highly purified water prepared from previously distilled water. Other buffers were prepared in deionized distilled water. Synthetic methods Standard chemical procedures as previously described were used (Baldwin et al., 1986). Melting points were recorded on a Buchi 510 apparatus and are uncorrected. I.r. spectra were recorded on a Perkin-Elmer model 681 spectrophotometer (s = strong, m = medium, w = weak, b = broad). 1H-n.m.r. spectra were either recorded at 300 MHz on a Bruker WH 300 NMR spectrometer or at 500 MHz on a Bruker AM 500 spectrometer. 1987

833

Cephalosporium deacetoxycephalosporin C synthetase/hydroxylase Mass spectra in the electron-impact mode or chemicalionization mode were recorded on a VG Micromass 16F spectrometer. Samples requiring desorption chemical ionization or fast atom bombardment were run on VG Micromass 30F or ZAB IF spectrometers.

(2S,5R,6R)-l-Aza-3,3'-dimethyl-6-(5R)-5-p-nitrobenzyl-

oxycarbonyl-5-p-nitrobenzyloxycarbonylaminopentan-

amidol-7-oxo-4-thiabicyclo[3,2,0heptane-2-carboxylic

acid p-nitrobenzyl ester (7) To a solution of 6-aminopenicillanic acid p-nitrobenzyl ester (6.60 g, 18.8 mmol) in dichloromethane (100 ml) was added compound 6 (Baldwin et al., 1980) (8.93 g, 18.8 mmol) and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (4.64 g, 18.8 mmol). The solution was stirred at 20 °C for 24 h and then evaporated to dryness. The residue was dissolved in ethyl acetate (200 ml), washed with 1 M-HCl (100 ml), 0.6 M-NaHCO3 (100 ml) and satd. NaCl (100 ml), dried (over Na2SO4), filtered and evaporated to dryness. Chromatography [flash silica, with dichloromethane/ethyl acetate (1: 0 to 1: 1, v/v)] gave compound 7 (9.60 g, 63% yield). T.l.c. (ethyl acetate/ hexane (1: 1, v/v)] RF 0.4. M.s. (electron impact) 808 (M+). Analysis: Found: C, 53.33; H, 4.69; N, 9.99; S, 3.89; calc. for C3,H36N6O,4S: C, 53.46; H, 4.45; N, 10.34; S, 3.90%. 'H n.m.r. & (p.p.m.) (300 MHz, [2H]chloroform) 1.43 (3H, s, CH3), 1.62 (3H, s, CH3), 1.69-1.95 (4H, m, CH2CH2CH2CO), 4.38-4.46 (1H, m, NCHCH2), 4.47 (1H, s, 2-H), 5.20 (2H, approx. s, CH2Ar), 5.23-5.36 (4H, m, 2 x CH2Ar), 5.52 (1H, d, J = 4.0 Hz, 5-H), 5.69 (1H, dd, J = 6.0 and 4.0 Hz, 6-H), 6.27 (1H, d, J = 9.0 Hz, NH), 7.47-7.58 (7H, m, 6 x Ar-H+NH) and 8.18-8.29 (6H, m, 6 x Ar-H). [a]2D + 86.60 (c = 1 in chloroform). I.r. (chloroform) 3440 (m), 1790 (s) (C=O, ,B-lactam), 1730 (s) (C=O, ester), 1690 (s) (C=O, amide), 1610 (s), 1525 (m), 1510 (m) and 1360

(s) (C-NO2) cm-'. (2S, 5R, 6R)-l-Aza-3,3'-dimethyl-6-(5R)-5-p-nitrobenzyloxycarbonyl-5-p-nitrobenzyloxycarbonylaminopentanamidol-7-oxo 4thiabicyclol3,2,01heptane-2carboxylic acid p-nitrobenzyl ester 0-sulphoxide (8) To a solution of compound 7 (9.21 g, 11.4 mmol) in dichloromethane (50 ml) at 0 °C was added dropwise a solution of m-chloroperbenzoic acid (2.04 g, 14.8 mmol) in dichloromethane (50 ml). The reaction mixture was stirred at 0 °C for 45 min and at 20 °C for 1 h. The organic solution was then washed with 2.0 MNaHSO3 (2 x 50 ml) and satd. NaCl (50 ml), dried (over Na2SO4) and evaporated to dryness. Chromatography [flash silica, with ethyl acetate/light petroleum (b.p. 40-60 °C) (0: 1 to 3: 1, v/v)] gave compound 8 (8.80 g, 94% yield) as a foam. T.l.c. [ethyl acetate/dichloromethane (3:2, v/v)] RF 0.2. Analysis: Found C, 50.30; H,

4.35; N, 9.43; calc. for C36H3,N6015S,2H20: C, 50.23; H, 4.18; N, 9.76%. 'H n.m.r. 6 (p.p.m.) (300 MHz, [2H]chloroform) 1.16 (3H, s, a-CH3), 1.69 (3H, s, fl-CH3), 1.74-1.93 (4H, m, CH2CH2CH2CO), 2.23-2.29 (2H, m, CH2CO), 4.38-4.44 (1H, m, NCHCH2),4.70 (1H, s, 2-H), 5.04 (1H, d, J = 4.5 Hz, 5-H), 5.20 (2H, approx. s, CH2Ar), 5.27 (2H, approx. s, CH2Ar), 5.34 (2H, approx. s, CH2Ar), 5.67 (1H, d, J = 8 Hz, NH), 6.05 (1H, dd, J - 10 and 4.5 Hz, 6-H), 7.01 (1H, d, J - 10 Hz, NH), 7.49-7.59 (6H, m, 6 x Ar-H) and 8.19-8.29 (6H, m, 6xAr-H). [a]2 + 85.80 (c = 1 in chloroform). I.r.

Vol. 245

(chloroform) 1800 (s) (C=O, fl-lactam), 1750 (s) (C=O, ester), 1730 (s) and 1680 (s) cm-1. Penicillin N /I-sulphoxide (4) To a solution of compound 8 (71 mg, 0.091 mmol) in tetrahydrofuran (10 ml) were added water (10 ml) and NaHCO3 (7.8 mg, 1 equivalent). The resultant solution was hydrogenated at 0.1 MPa (1 atm gauge) over 10% Pd on charcoal (100 mg) for 3 h. The solution was then filtered (Celite), washing with water (10 ml), and the filtrate was washed with ethyl acetate (2 x 10 ml). The aqueous layer was then freeze-dried to give crude compound 4. Chromatography (reverse-phase h.p.l.c., packing material Zorbax ODS Cl., eluent 25 mmNH4HCO3, retention time 20 min) gave pure (by 'H n.m.r.) compound 4 as the ammonium salt (25 mg, 73 % yield). 'H n.m.r. 4 (p.p.m.) (300 MHz, 2H20) 1.11 (3H, s, a-CH3), 1.50 (3H, s, fl-CH3), 1.45-1.59 and 1.631.77 (4H, 2 x m, CH2CH2CH2CO), 2.19-2.23 (2H, m, CH2CH2CO), 3.60-3.33 (1H, m, NCHCH2), 4.31 (1H, s, 2-H) and 5.18 and 5.67(2 x 1H, 2 x d, J = 4.5 Hz, 5-H+6-H). The stereochemistry of the sulphoxide was established by nuclear Overhauser spectroscopy in an analogous manner to that used for isopenicillin N fl-sulphoxide (Bahadur et al., 1981). (2R, 3R)-2-(Benzothiazole-2-dithio)-1-[(lR)-1-isoprop1-enylacetic acid p-nitrobenzyl esterl-3-I(5R)-5p-nitrobenzyloxycarbonyl-5-p-nitrobenzyloxycarbonylaminopentanamidolazetidin-4-one (9) A solution of compound 8 (8.80 g, 8.92 mmol) in toluene (50 ml) was treated with 2-mercaptobenzothiazole (1.50 g, 8.9 mmol) and refluxed for 4 h. The solution was evaporated to dryness, after which chromatography [flash silica, with ethyl acetate/light petroleum (b.p. 40-60 °C) (0:1 to 3:1, v/v)] gave compound 9 as a foam (6.37 g, 62% yield). T.l.c. [ethyl acetate/light petroleum (b.p. 40-60 °C) (3:2, v/v)] RF 0.3. 'H n.m.r. 4 (p.p.m.) (300 MHz, [2H]chloroform) 1.72-1.85 (4H, m, CH2CH2CH2CO), 1.93 (3H, s, CH3), 2.29-2.41 (2H, m, CH2CH2CO), 4.38-4.45 (1H, m, NHCHCH2), 5.01 and 5.07 (2H, 2 x s, C=CH2), 5.17 (2H, approx. s, CH2Ar), 5.19 (2H, approx. s, CH2Ar), 5.21 (2H, approx. s, CH2Ar), 5.22 (1H, s, NCHCC=CH2), 5.37 (1H, dd, J = 8.0 and 5.0 Hz, 3-H), 5.51 (1H, d, J = 5.0 Hz, 2-H), 5.82 (1H, d, J = 8.0 Hz, NH), 7.08 (1H, d, J = 8.0 Hz, NH), 7.29-7.50 (8H, m, 8 x Ar-H), 7.73-7.85 (2H, m, 2 x Ar-H) and 8.10-8.22 (6H, m, 6 x Ar-H). (2S, 3R, 5R, 6R)-l-Aza-3-chloroacetoxymethyl-3-

methyl-6[(5R)-5-p-nitrobenzyloxycarbonyl-5-p-nitrobenzyloxycarbonylaminopentanamidol-7-oxo-4-thia-

bicyclo[3,2,Ojheptane-2-carboxylic acid p-nitrobenzyl ester (10) and (2R,3S,6R,7R)-1-aza-3-chloro-

acetoxymethyl-3-methyl-7-[(5R)-5-p-nitrobenzyloxy-

carbonyl-5-p-nitrobenzyloxycarbonylaminopentanamidol8-oxo-5-thiabicyclo[4,2,Ojoctane-2-carboxylic acid p-

nitrobenzyl ester (11) A solution of compound 9 (4.00 g, 4.11 mmol) in dichloromethane (200 ml) was treated with silver chloroacetate (1.37 g, 8.22 mmol) and chloroacetic acid (17.47 g, 184 mmol) and stirred for 3 h at 20 'C. The reaction mixture was filtered (Celite), washing with dichloromethane (100 ml). The filtrate was washed with satd. NaHCO3 (50 ml), water (50 ml) and satd. NaCl (50 ml), dried (over MgSO4) and evaporated to

834

dryness. Chromatography [preparative t.l.c., with ethyl acetate/light petroleum (b.p. 40-60 °C) (3:2, v/v), three times] gave compounds 10 and 11. For compound 10 (654 mg, 18% yield): t.l.c. [ethyl acetate/light petroleum (b.p. 40-60 °C) (3:2, v/v)] RF 0.30. Analysis: Found: C, 50.18; H, 4.47; N, 9.25; S, 3.01; calc. for C38H37CIN6 16S: C, 50.66; H, 4.11; N, 9.33; S, 3.55%. 1H n.m.r. 6 (p.p.m.) (300 MHz, [2H]chloroform) 1.46 (3H, s, CH3), 1.64-1.96 (4H, m, CH2CH2CH2CO), 2.33-2.42 (2H, m, CH2CH2CO), 3.88, 4.58 (2H, ABq, J = 11.5 Hz, CH2OCOCH2C1), 4.16 (2H, s, CH2Cl), 4.46-4.49 (1H, m, CHCH2CH2), 4. 68 (1H, s, 2-H), 5.21-5.39 (6H, m, 4 x CH2Ar), 5.61 (1H, d,J = 4Hz, 5-H), 5.63 (1H, obscured d, NH), 5.73 (1H, dd, J = 9.5 and 4 Hz, 6-H), 6.83 (1H, d, J = 9.5 Hz, NH), 7.49-7.59 (6H, m, 6 x Ar-H) and 8.63-8.73 (6H, m, 6 x Ar-H). [z]20 +64.5° (c = 1 in chloroform). I.r. (chloroform) 1790 (s) (C=O, ,f-lactam), 1750 (s) (C=O, ester), 1690 (s) (C=O, amide), 1610 (m) and 1360 (s) (C-NO2) cm-1. For compound 11 (810 mg, 22% yield): t.l.c. [ethyl acetate/light petroleum (b.p. 40-60 °C) (3:2, v/v)] RF 0.40. 'H n.m.r. 6 (p.p.m.) (300 MHz, [2H]chloroform) 1.55 (3H, s, CH.3), 1.71-1.91 (4H, m, CH2CH2CH2CO), 2.23-2.35 (2H, m, CH2CH2CO), 3.30, 4.04 (2H, ABq, J= 15.5 Hz, 4-H), 4.06, 4.16 (2H, ABq, J= 11.5 Hz, CH2Cl), 4.87 (1H, s, 4-H), 4.89 (1H, d, J = 4.5 Hz, 6-H), 5.20 (2H, approx. s, CH2Ar), 5.27 (2H, s, CH2Ar), 5.30 (2H, s, CH2Ar), 5.60 (1H, dd, J = 9.5 and 4.5 Hz, 7-H), 5.68 (1H, d, J = 8 Hz, NH), 6.50 (1H, d, J = 9.5 Hz, NH), 7.45-7.53 (6H, m, 6 x Ar-H) and 8.14-8.43 (6H, m, 6xAr-H).

(2S,3R,5R,6R)-1-Aza-3-hydroxymethyl-3-

methyl-6-(5R)-5-p-nitrobenzyloxycarbonyl-5-p-nitrobenzyloxycarbonylaminopentanamidol-7-oxo4-

thiabicyclo[3,2,O1heptane-2-carboxylic acid p-nitrobenzyl ester (12) A solution of compound 10 (273 mg, 0.30 mmol) in acetone (10 ml) was treated with KI (151 mg, 0.91 mmol) and stirred for 2 h at 20 'C. NN'-Di-n-butylthiourea (284 mg, 1.52 mmol) was added and the reaction mixture was stirred for a further 20 h, after which it was evaporated to dryness (Kamiya et al., 1973). Chromatography (Sephadex LH-20, with ethanol) gave compound 12, as a white foam (72 mg, 29% yield). Analysis: Found: C, 50.19; H, 4.37; N, 9.58; calc. for C36H36N6015S,2H20: C, 50.23; H, 4.18; N, 9.76%. M.s. (chemical ionization) 825 (M++H). 'H n.m.r. 6 (p.p.m.) (300 MHz, [2H]chloroform) 1.38 (3H, s, CH3), 1.51-1.94 (4H, m, CH2,CH2CH2CO), 2.25-2.64 (2H, m, CH2CH2CO), 3.54, 3.74(2H,ABq,J= 11.5 Hz, CH2OH),4.39-4.51 (lH,m, CHCH2CH2), 4.73 (1H, s, 2-H), 5.22-5.33 (6H, m, 3 x CH2Ar), 5.60-5.74 (2H, m, 5-H+6-H), 6.75 (1H, d, J = 9 Hz, NH), 7.50-7.63 (7H, m, 6 x Ar-H+ NH) and 8.17-8.33 (6H, m, 6xAr-H). [c]20 +64.70 (c = 1, in chloroform). I.r. (chloroform) 1790 (s) (C=O, ,-lactam), 1745 (s) (C=O, ester), 1680 (s) (C=O, amide), 1615 (m) and 1365 (s) (C-NO2).

(2S,3R,5R,6R)-1-Aza-6[(5R)-5-carboxypentanamido-

3-hydroxymethyl-3-methyl-7-oxo 4 thiabicyclo[3,2,01heptane-2-carboxylate (5) To a solution of compound 12 (9 mg, 0.01 mmol) in tetrahydrofuran (2 ml) was added a solution of

J. E. Baldwin and others

NaHCO3 (1 mg, 0.01 mmol) in water (2 ml). The resultant solution was hydrogenated at 0.1 MPa (1 atm gauge) over 10% Pd on charcoal (25 mg) for 12 min. The solution was then filtered (Celite), washing with water (5 ml), and the filtrate was washed with ethyl acetate (2 x 10 ml). The aqueous layer was then evaporated to dryness to give crude compound 5. Attempted purification of compound 5 (reverse-phase h.p.l.c., packing material 1sBondapak Cl,, eluent 10 mM-NH4HCO3) resulted in decomposition of compound 5 and the isolation of compound 13, which isomerized on standing in aqueous solution. For compound 5: partial 'H n.m.r. 6 (p.p.m.) (500 MHz, 2H20) 5.31 and 5.48 (2H, 2 x d, J = 4.0 Hz, 5-H+ 6-H). Compound 5 showed antibacterial activity against S. aureus N.C.T.C. 6571. For compound 13: 'H n.m.r. 6 (p.p.m.) (500 MHz, 2H20) 1.11 (3H, s, CH3), 1.51-1.86 (4H, m, CH2CH2CH2CO), 2.19-2.25 (2H, m, CH2CH2CO), 3.57, 3.72 (2H, ABq, J = 11.0 Hz, CH2OH), 4.09 (1H, d, J= 8.5Hz, CHS) and 4.80 (1H, d, J= 8.5 Hz, NHCHCHS). M.s. (fast atom bombardment) 394 (M++H) and 416 (M++Na).

(2S,4S,6R,7R)-1-Aza4-methyl-7-1(5R)-5-p-nitrobenzyloxycarbonyl-5-p-nitrobenzyloxycarbonylaminopentan-

amidol-8-oxo-5-thiatricyclo-14,2,0,02'41octane-2-

carboxylic acid p-nitrobenzyl ester (17) To a solution of compound 15 (300 mg, 0.64 mmol) in dichloromethane (5 ml) and NN-dimethylaniline (0.30 ml) at 0 °C was added PCI5 (180 mg, 0.87 mmol), and the mixture was allowed to warm to 20 °C over 1 h. The mixture was then cooled to 0 °C and treated with ethanol (2.0 ml), allowed to warm to 20 °C over 1 h, treated with water (40 ml) and stirred for 15 min. The solution was neutralized to pH 7 (1 M-NaOH) and extracted with ethyl acetate (3 x 100 ml). The organic extracts were combined, dried (over Na2SO4), filtered and evaporated to dryness. Chromatography [flash silica, with dichloromethane/ethyl acetate (1:0 to 0:1, v/v)] gave partially purified compound 16 (205 mg, 95% yield). This material was dissolved in diethyl ether (5 ml) and ethyl acetate (5 ml) and treated with anhydrous toluene-p-sulphonic acid (114 mg, 0.66 mmol) in diethyl ether/ethyl acetate (1: 1, v/v) (10 ml). The resultant precipitate was filtered off, washed with diethyl ether (3 x 20 ml) and partitioned between ethyl acetate (150 ml) and 0.6 M-NaHCO3 (50 ml). The organic layer was separated, re-extracted with satd. NaCl (2 x 50 ml), dried (over Na2SO4), filtered and evaporated to dryness, to yield compound 16 as an oil (60 mg, 26% yield), 'H n.m.r. 6 (p.p.m.) (300 MHz, [2H]chloroform) 1.42 (3H, s, CH3), 1.80-2.00 (2H, m, 3-H), 4.15 (1H, d, J = 5.0 Hz, 7-H), 5.05 (2H, approx. s, CH2Ar), 5.95 (IH, d, J = 5.0 Hz, 6-H), 7.20-7.35 (2H, m, 2 x Ar-H) and 7.95-8.10 (2H, m, 2 x Ar-H). The oil was dissolved in dichloromethane (10 ml), 2-ethoxy- 1 -ethoxycarbonyl- 1,2-dihydroquinoline (51 mg, 0.21 mmol) and compound 6 (97 mg, 0.20 mmol) were added and the solution was stirred under argon for 36 h. The solution was evaporated to dryness, redissolved in ethyl acetate (100 ml), washed with 2 M-HCI (50 ml), 0.6 M-NaHCO3 (50 ml) and satd. NaCl (50 ml), dried (over Na2SO4), filtered and evaporated to dryness. Chromatography [flash silica, with dichloromethane/ ethyl acetate (1:0 to 1:4, v/v) gave compound 17 as a foam (120 mg, 23 % yield), m.p. 76-77 'C. T.l.c. [ethyl 1987

Cephalosporium deacetoxycephalosporin C synthetase/hydroxylase

835

24

1.2 ca E

,u

>

.

50 O

Ca'

-

Ecm .> :

0

Wx v)

-

40 c

4cC

_

CM

._ -r,0 n

.0 %

0

Cm co _,

< 0,

Q0,.

0

20

40

60 Time (h)

80

100

20.: 10 ° x

0

-

Fig. 1. Growth profile of C. acremonium and time course for enzyme and andbiotdc production C. acremonium C.O. 728 was grown in standard medium as described in the text. El, A550; A, isopenicillin N synthetase specific activity; 0, expandase specific activity; *, cephalosporin C; A, penicillin N. For details see the text.

acetate/dichloromethane, 1: 1, v/v) RF 0.45. Analysis: Found: C, 53.38; H, 4.31; N, 10.28; S, 4.17; calc. for C36H34N6014S: C, 53.60; H, 4.25; N, 10.42; S, 3.97%. M.s. (field desorption) 806 (M+). 'H n.m.r. 6 (p.p.m.) (300 MHz, [2H]chloroform) 1.64 (3H, s, CH3), 1.65-2.00 (4H, m, CH2CH2CH2CO), 2.10-2.24 (2H, ABq, J = 12 Hz, 3-H), 2.26 (2H, approx. t, J = 7.0 Hz, CH2CONH), 4.40-4.42 (1H, m, CHCH2CH2CH2CO), 5.21, 5.23, 5.31 (6H, 3 x approx. s, 3 x CH2Ar), 5.49 (1H, dd, J = 8.0 and 4.0 Hz, 7-H), 6.19 (1H, d, J = 8.0 Hz, NH), 6.19 (1H, d, J= 4.0 Hz, 6-H), 6.44 (1H, d, J = 8.0 Hz, NH), 7.49-7.55 and 8.17-8.26 (12H, 2 x m, 12 x Ar-H). 13C n.m.r. a (p.p.m.) (62.5 MHz) 20.0 (q, CH3), 20.8 (t, CH2CH2CH2CO), 31.5 (t, CH2CH2CH2CO), 34.8 (t, CH2CO), 41.5 (t, 3-C), 52.0, 55.3 (2 x s, 2-C+4-C), 53.7, 57.7 (2 x d, 7-C+CHCH2 CH2CH2), 65.5, 65.7, 66.2 (3 x t, CH2Ar), 78.4 (d, 6-C), 123.8, 123.9, 124.0 (3xd, m-Ar-C), 128.1, 128.5, 128.6 (3 x d, o-Ar-C), 142.2, 142.3, 143.6 (3 x s, ipso-Ar-C), 147.7, 147.9, 147.9 (3 x s, p-Ar-C), 155.6, 167.3, 171.6, 171.7 and 173.5 (5 x s, CO). U.v. Amax (acetonitrile) 258nm (e= 34300M-1 cm-'). [a]2. +114° (c= 1 in chloroform). I.r. (chloroform) 3690 (m), 1795 (m) (C=O, ,-lactam), 1730 (s) (C=O, ester), 1685 (s) (C=O, amide), 1610 (m), 1420 (s), 1360 (s) (C-NO2), 1060 (m), 1015 (m) and 930 (s) cm-'.

(2S,4S,6R,7R)-l-Aza-7-[(5R)-5-carboxypentanamidolj4methyl-8-oxo-5-thiatricyclo-14,2,0,02,4]octane-2-

carboxylate (14) To a solution of compound 17 (65 mg, 0.08 mmol) in tetrahydrofuran (10 ml) was added 8.20 ml of a solution of NaHCO3 in water (8.3 mg in 10 ml). The resultant solution was hydrogenated at 0.1 MPa (1 atm gauge) over 10% Pd on charcoal (50 mg) for 1 h. The solution was then filtered (Celite), washing with water (10 ml), and the filtrate was washed with ethyl acetate (2 x 25 ml). The aqueous layer was then freeze-dried to give compound 14 (27 mg, 88% yield). 'H n.m.r. 6 (p.p.m.) (300 MHz, 2H20) 1.42 (3H, s, CH3), 1.42-1.71 (4H, m, CH2CH2CH2CO), 1.71, 1.91 (2H, ABq, J= 12 Hz, 4-H), 2.19 (2H, approx. t, J= 7.0 Hz, CH2CH2CO), 3.50-3.54 (1H, m, CHCH2CH2), 5.10 (1H, d, J=4.0Hz, 7-H), 6.04 (1H, d, J=4.0Hz, Vol. 245

6-H). '3C n.m.r. 6 (p.p.m.) (62.5 MHz, 2H20) 20.3 (q, CH3), 21.8 (t, CH2CH2CH2CO), 30.7 (t, CH2CH2CH2CO), 35.4 (t, CH2CON), 40.4 (t, 3-C), 53.4, 55.1 (2 x s, 2-C+4-C), 55.4 58.3 (2 x d, CHCH2CH2+7-C), 77.9 (d, 6-C), 174.7, 175.4, 175.8 and 176.8 (4xs, 4xCO). I.r. (Nujol) 3650-2300 (b) (OH), 1770 (s) (C=O, fl-lactam) and 1600 (b) cm-'. RESULTS Growth profile and time course for enzyme and antibiotic production Fig. 1 shows the growth profile of C. acremonium C.O. 728, together with the time course for production of expandase activity, isopenicillin N synthetase activity, penicillin N (1) and cephalosporin C, in standard medium [2.7% (w/v) glucose/3.6% (w/v) sucrose] with asparagine (0.75%) as the nitrogen source. Raising the concentration of asparagine to 1.2% resulted in lower specific activity of the crude enzyme preparations. Enzyme purification Initial stages. All steps were carried out at 4 'C. Protamine sulphate was added to the cell-free extract to a final concentration of 0.5%. After centrifugation at 12000 g for 30 min, (NH4)2SO4 was added to the supernatant to a final concentration of 55% saturation. After equilibration and centrifugation (12000 g for 30 min), the supernatant was made up to 75% saturation with (NH4)2SO4, and the precipitate was collected by centrifugation at 12 000 g for 30 min. This material was redissolved in the extraction buffer (Pang et al., 1984), and loaded on to a Sephadex G-75 gel-filtration column (130 cm x 5 cm) equilibrated with extraction buffer. After elution overnight at 4°C, fractions were tested for expandase activity by bioassay. Active fractions were pooled and applied (flow rate 24 ml/h) to a column of DEAE-Sepharose (fast flow) (10 cm x 5 cm), pre-equilibrated with extraction buffer, at 4 'C. Expandase was eluted with a linear gradient of 0.05-0.3 M-NaCl in buffer, over 300 ml, at a flow rate of 24 ml/h. Active fractions, tested by bioassay, were pooled. The product (crude

J. E. Baldwin and others

836 160 1AA

140

(a)

2.8

0.7

280

(b)

-

120

2.4

240

0.6

2.0

200

1.6 Go (0

-

3

E

0

160 >

0 0

120 ,

._

1.2

0.8 -'

40

0.4[o-

20

C

0

40

0.1 0

200

400

600

800

0

80

_JO 4J 8

2-

200

0

400

a

600

C

a. -5 42 coX r

--w

x

0

--4

200

400 600 Volume (ml)

800

0

200 400 Volume (ml)

0

"

600

Fig. 2. Purification of expandase from C. acremoniwm (a) Dye-ligand chromatography on Matrex Gel Red A. 0, A280; 0, expandase activity; A, conductivity. (b) Hydrophobic chromatography on phenyl-Sepharose (fast flow). O, A280; 0, expandase activity; A, conductivity. For details see the text.

enzyme) was used for study of some of the properties of the enzyme described below.

Dye-ligand chromatography, ion-exchange f.p.l.c., gel-filtration f.p.l.c. and hydrophobic chromatography. For purification to near homogeneity, as judged by SDS/polyacrylamide-gel electrophoresis, pooled active fractions after Sephadex G-75 chromatography were applied (flow rate 10 ml/h) to a column (30 cm x 2.5 cm) of Matrex Gel Red A (Procion Red HE3B-agarose). In preliminary experiments Procion

Red (0.1 mM) was found to inhibit reversibly expandase activity by virtually 100%. The column was washed with 350 ml of extraction buffer containing DTT (2 mM) at a flow rate of 24 ml/h, and enzyme was eluted with a linear gradient of 0-2.0 M-KCI in buffer over 500 ml, at the same flow rate, as shown in Fig. 2(a). Active fractions were free from isopenicillin N synthetase activity, since isopenicillin N synthetase did not bind to Matrex Gel Red A. Active fractions were pooled, exchanged into 20 mM-Tris/HCI buffer, pH 8.0, containing DTT (2 mM), and applied (flow rate

Table 1. Purification of deacetoxycephalosporin C synthetase (expandase) from C. acremoniwn For details see the text. Values of percentage yield are calculated relative to step 1.

Volume

103 x Specific

activity

Yield (%)

Purification (fold)

(mg/ml)

(units/ml)

(units)

(units/mg)

109

5.90

8.26

0.9

1.4

100

39

0.90

4.32

0.17

4.8

19

0.14

2.59

0.03

18.5

3.3

13.2

0.05

0.81

0.03

16.1

3.3

11.5

0.10

3.85

0.004

38.5

0.4

27.5

Step

(ml)

1. Sephadex G-75

chromatography 2. Dye-ligand chromatography on Matrex Gel Red A 3. Ion-exchange chromatography on Mono Q f.p.l.c. 3a. Hydrophobic chromatography on phenyl-Sepharose (fast flow) after

Total Concn. of 103 x Expandase expandase protein activity activity

1.15 37.0

3.4

Matrex Gel Red A

chromatography 4. Gel filtration on Superose 12 f.p.l.c.

1.03

1987

837

Cephalosporium deacetoxycephalosporin C synthetase/hydroxylase 70 r 60 I E c 50

.2

.> 40

_-

X0. 30

_

Q

X

x20

o

10o

5

6

7

8 pH

9

10

11

Fig. 3. Effect of pH on partially purified expandase Buffers were as follows: 0, Tris/HCl; [, sodium phosphate; A, diethanolamine; *, Bistris/HCl. For details see the text.

8 ml/min) to a Mono Q 16/10 strong anion-exchange column for f.p.l.c. Enzyme was eluted with a linear gradient of 80-180 mM-NaCl in 20 mM-Tris/HCl buffer, pH 8.0, containing DTT (2 mM), at the same flow rate. Active fractions were pooled, desalted, concentrated and applied (flow rate 0.25 ml/min) to a Superose 12 f.p.l.c. gel-filtration column. Enzyme was eluted with 0.1 M-Tris/HCl buffer, pH 8.0, containing DTT (2 mM). Active fractions were pooled and concentrated. In alternative experiments, active fractions after Procion Red chromatography were pooled, concentrated, made 1.2 M with respect to (NH4)2SO4, in 50 mM-Tris/HCl buffer, pH 7.4, containing DTT (2 mM) and applied to a column (28 cm x 1.5 cm) of phenyl-Sepharose (fast flow). Enzyme was eluted (flow rate 24 ml/h) with a linear gradient of 1.2-0 M-(NH4)2SO4 in pH 7.4 buffer (Fig. 2b). Active fractions were pooled and concentrated. Details of a typical enzyme purification are shown in Table 1. At each stage fractions with less than 30% of maximum activity were discarded. A similar final specific activity of 0.04 unit/mg was obtained by chromatography on DEAE-Sepharose followed by DEAE-TSK 545 h.p.l.c.

Enzymic properties The effect of pH on partially purified expandase activity in 0.1 M buffers, judged by bioassay, is shown in Fig. 3. Sodium phosphate and Bistris buffers (0.1 M) inhibited activity. Similar results were obtained in 50 mM buffers. Ascorbate was essential for activity, and in the presence of ascorbate DTT caused a 30% increase in activity up to 1 mM-DTT. D-Isoascorbate had the same effect as its isomer. However, the effect of DTT was approx. 25% greater with 'reduced' enzyme, where enzyme had been exposed to DTT (1 mM) throughout the purification procedure, than with 'oxidized' enzyme, where 'reduced' enzyme had been exchanged into buffer without DTT and incubated at 27 °C for 90 min an orbital shaker before bioassay. Partially purified enzyme was stable for over 4 weeks at -70 °C provided that DTT (1 mM) was present. At 4 °C the half-life of expandase activity was found to be 48 h in the presence of DTT (1 mM). Addition of ascorbic acid (1 mM), FeSO4 (0.04 mM), 2-oxoglutarate (0.8 mM), (NH4)2SO4 (100 mM) or bovine liver catalase (40000 units/ml) to the stored enzyme in the absence of DTT had little effect on stability. 2-Oxoglutarate was essential for activity '(Km 0.04 mm by bioassay), and could not be replaced by 2-oxoadipate or pyruvate. The addition of ATP was found not to enhance activity, in agreement with Scheidegger et al. (1984). Addition of NH4HCO3 (100-500 mM) to the bioassay mixture completely inhibited expandase activity, whereas NH4Cl and (NH4)2S04 caused 2.8-fold enhancement of activity at 500 mm. NaCl, Na2SO4 and KCI had little effect on activity. Expandase activity was inhibited by the thiol-quenching reagent N-ethylmaleimide (0.1 mM). The metal-ion-chelating reagent bathophenanthroline (4,7-diphenyl-1,10-phenanthroline) inhibited activity by 50% at 0.02 mm, whereas the non-chelating 4',7'-isomer had no effect. Chemical syntheses Oxidation of tri-protected penicillin N (7) (Baldwin et al., 1980) (m-chloroperbenzoic acid, 0 °C, dichloromethane) gave exclusively the ,-sulphoxide (8) (94%), which was deprotected (H2, Pd/charcoal) to give compound 4 (Scheme 2). The synthesis of compound 5 was achieved by utilizing mild interconversions established in model 0 Ho

PNB02CNH

COH

HC0PN S CO2 PNB

H-~z~C°

PNBO2CHN

CON

COHN B°

U2 r'F'NB

8

PNB = 4-O2NC6H4CH2 H

COOH e1 O2 CO2PNB 2

6

Scheme 2.

Vol. 245

0

.

7

PNB02CHN

H3N *1-CO H N

;

C02PNB

O0

+

I

4

v

1'

CO2 H

J. E. Baldwin and others

838

PNBO2CNHt 8

-

PNBO2CHN _

COHNNcOH

C02PNB

k

1

COHN

C

,

t

t H C02PNB

H20COCH2CI

C02PNB

CO2PNB

10

9

PNB02CHNg H

COHN

CO2PNB

S OCOCH2CI

N

CO2PNB 11

Scheme 3. PNB02CHN

CONH+ -~~~~H~r~ 2 CCHN N[o C2PNB

H2H3N C0HN2H20H C~NOHN SCHH C02PNB 0 CO2 H

10 10

H

5

12

Scheme 4.

studies (Baldwin & Singh, 1979). Thus treatment of the disulphide (9), prepared by the procedure of Kamiya et al. (1973) from compound 7, with silver chloroacetate gave a mixture of the penam (10) (18%) and the cepham (11) (22%) (Scheme 3). Reaction of compound 10 with KI followed by NN'-di-n-butylthiourea gave compound 12. Deprotection (H2, Pd/charcoal) gave the desired 3,8-hydroxypenam (5) (positive bioassay against S. aureus N.C.T.C. 6571) (Scheme 4). However, compound 5 was found [by 'H n.m.r. (500 MHz)] to be unstable in aqueous solution and rapidly decomposed to give the penicilloate (13), which equilibrated (at C-2) upon standing in 2H20. H3N H

The tricyclic penam (14) was also synthesized for evaluation as a substrate for the expandase reaction. Thus removal of the phenylacetyl side chain of compound 15 (NN'-dimethylaniline/PCl5) gave the amine (16), which was coupled with diprotected D-a-aminoadipic acid (6) to give compound 17. Deprotection (H2, Pd/charcoal) gave the desired tricyclic penam (14) (Scheme 5).

Substrate specificity of expandase Incubation of compounds 4 and 5 (in the case of the latter, immediately after deprotection of compound 12) with the crude enzyme (under conditions that

C

CH

C2-

02C

S CHNH H2N1_C2 CO2H

13 C6

H5CH2COHN

H2N

S

PNBO2CHN-

S

COHN

CO2PNB 0

CO2PNB 15

C02 PN B

C02 PNB

16

H3N

v

54-10%

17

COHN

CO2C02

5

s' %C 002 H

14

Scheme 5.

1987

Cephalosporium deacetoxycephalosporin C synthetase/hydroxylase

839

100

(i) 80

y

E

60 >

coN

200

400

6,*

40O

0,,

20 0

0

0

600

w

ww 2 '__E

-

1 x

Ox

0

200

400 Volume (ml)

600

800

0.05

z

0)

.0

0.04

(ii)

0.03

C

X,

o

0.02

0oo1 L

Fig. 4. Ion-exchange chromatography on DEAE-Sepharose (fast flow) of material after Sephadex G-75 gel filtration (see Table 1)

0, A280; 0, expandase activity, measured by bioassay; A, conductivity. For details see the text. X and Y are peaks analysed for expandase and hydroxylase activities by h.p.l.c. (see Fig. 5).

L

oL

0.05

(iii)

0.04

0.03

permitted conversion of compound 1 into compounds 2 and 3 at a 1 mg testing level) did not give rise to cephem products, judged by 1H n.m.r. (500 MHz) or bioassay as described above. The tricyclic penam 14 was not found to be a substrate for expandase activity. However, in a competitive experiment with an equimolar amount of compound 1 (under standard incubation conditions), it was found to be a potent reversible inhibitor of the transformation of compound 1 into compound 2, causing over 90% inhibition at a concentration of 40 ,UM. Deacetoxycephalosporin C hydroxylase activity Throughout the purification procedure fractions were analysed for both expandase and hydroxylase activities by using h.p.l.c. or t.l.c. methods. Figs. 4 and 5 show analysis of both activities after DEAE-Sepharose (fast flow) ion-exchange chromatography. Two peaks of expandase activity were separated on DEAE-Sepharose (Fig. 4). Both peaks contained hydroxylase activity, as judged by h.p.l.c (Fig. 5) and t.l.c. assays. Fig. 6 shows a typical t.l.c. bioassay analysis of hydroxylase activity in fractions eluted from phenyl-Sepharose (fast flow) chromatography, where elution of hydroxylase activity corresponded exactly to elution of expandase activity, as judged by the standard bioassay. In further experiments, enzyme after the Sephadex

0.02 . 0.01 0

0.05 r (iv)

text.

Vol. 245

0.04 I 0.03

F

0.02 I 0.01 I

A

D

0 c

0.05

(v)

0.04

0.03 I 0.02 1 0.01 I 0

A

L A

0.05

(vi)

Fig. 5. H.p.l.c. analysis of fractions from peaks X and Y after DEAE-Sepharose (fast flow) chromatography (see Fig. 4) (i) Standards [A, deacetylcephalosporin C (DAC); B, penicilloate; C, deacetoxycephalosporin C (DAOC); D, penicillin N]; (ii) blanks without substrates at t = 0; (iii) penicillin N as substrate at t = 0; (iv) penicillin N as substrate at t = 60 min; (v) DAOC as substrate at t = 0; (vi) DAOC as substrate at t = 60 min. For details see the

0

0.04 0.03 0.02 0.01 0

0

10

5

x

15 0 5 Volurme (ml)

10

Y

15

J. E. Baldwin and others

840 -i

LO

;r

ID -g

-

*

C

'14

CN c v;

0

qr

'g

M CII

0) M

Table 2. Amino acid composition of expandase/hydroxylase

co CV)

P-

M

For details see the text. - DAC

DAOC

-

Fig. 6. T.I.c. analysis of hydroxylase activity in fracdons after phenyl-Sepharose (fast flow) chromatography Fractions eluted from hydrophobic chromatography on phenyl-Sepharose (fast flow) (Fig. 2b) were analysed directly for hydroxylase activity by t.l.c. assay. L, Sample loaded onto column (after chromatography on Matrex Gel Red A); 36-46, fraction numbers (fraction volume 10 ml); S, DAC and DAOC standards. For details see the text.

c

u

-0

CM

°L)UL)

x

0,

C

Total

37 21 22 37 20 31 31 28 7 12 30 13 18 6 21 25

359

>.

..:. ... ..:

X66

-i

45

E -O

.30

:3 0, 0

- {s, *;

Asx (Asp + Asn) Thr Ser Glx (Glu + Gln) Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg

Composition

(residues/40000 Da)

polyacrylamide-gel electrophoresis. Gel filtration on Superose 12 f.p.l.c., by comparison with markers of known molecular mass, showed no evidence for enzyme polymerization in the native state.

*~~~~~~~~~~~~~~~~~~~~~~~~~.

i,

Amino acid

^ ;-; ^ ;Y };t

~~~~~17.2